The role of residues R97 and Y331 in modulating the pH optimum
of an insect b-glycosidase of family 1
Sandro R. Marana, Lu
´
cio M. F. Mendonc¸a, Eduardo H. P. Andrade, Walter R. Terra and Cle
´
lia Ferreira
Departamento de Bioquı
´
mica, Instituto de Quı
´
mica, Universidade de Sa
˜
o Paulo, Brazil
The activity of the digestive b-glycosidase from Spodoptera
frugiperda (Sfbgly50, pH optimum 6.2) depends on E399
(pK
a
¼ 4.9; catalytic nucleophile) and E187 (pK
a
¼ 7.5;
catalytic proton donor). Homology modelling of the
Sfbgly50 active site confirms that R97 and Y331 form
hydrogen bonds with E399. Site-directed mutagenesis
showed that the substitution of R97 by methionine or lysine
increased the E399 pK
a
by 0.6 or 0.8 units, respectively,
shifting the pH optima of these mutants to 6.5. The substi-
tution of Y331 by phenylalanine increased the pK
a

ing end of di- and/or oligosaccharides. According to the
CAZy website this family comprises 422 sequenced
b-glycosidases, of which the tertiary structure of 12 has
been determined. Together with families 2, 10, 17, 26, 30, 35,
39, 42, 51, 53, 59, 72, 79 and 86 family 1 forms clan A,
a group of families that shares structural and catalytic
similarities [1]. All b-glycosidases of family 1 present the
same tertiary structure [the (b/a)
8
barrel], they are configur-
ation-retaining glycosidases and their catalytic activity
depends on two glutamic acid residues, one positioned
after bstrand 4 and the other after b strand 7 [1]. These
glutamic acids are very close inside the active site (about
4.5 A
˚
apart) [2], and during the reaction the first glutamic
acid acts as proton donor, and the second acts as a
nucleophile. The catalytic nucleophile pK
a
is around 5.0 and
the catalytic proton donor pK
a
is around 7.0 [3–7].
Aplotofb-glycosidase activity vs. pH presents a bell
shape, indicating that in the pH optimum the catalytic
nucleophile is deprotonated and the catalytic proton donor
is protonated. Hence the branch of the curve below the pH
optimum is determined mainly by the ionization of the
catalytic nucleophile, whereas the catalytic ionization of the

¼ 4.9) and E187 (catalytic proton donor, pK
a
¼ 7.5).
Correspondence to S. R. Marana, Departamento de Bioquı
´
mica,
Instituto de Quı
´
mica, USP, CP 26077, Sa
˜
o Paulo, 05513–970, Brazil.
Fax: +55 11 30912186, E-mail:
Abbreviations: MU, 4-methylumbelliferyl; MUbglc, 4-methylumbel-
liferyl b-
D
-glucopyranoside; NPbglc, p-nitrophenyl, b-
D
-gluco-
pyranoside; Sfbgly50, digestive b-glycosidase (Mr 50 000) from
Spodoptera frugiperda; ES, enzyme substrate.
Enzyme:digestiveb-glycosidase from Spodoptera frugiperda
(b-
D
-glucoside glucohydrolase; EC 3.2.1.21; GenBank accession no.
AF052729).
(Received 18 July 2003, revised 25 September 2003,
accepted 21 October 2003)
Eur. J. Biochem. 270, 4866–4875 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03887.x
The effect of pH on Sfbgly50 activity is a typical bell-shaped
curve and the pH optimum is 6.2 [9].

a
values of the b-glycosidase catalytic glutamates still
remains to be determined and quantified.
To fill these gaps, residues Y331, R97 and E187 of
Sfbgly50 were replaced through site-directed mutagenesis by
phenylalanine (Y331F), methionine (R97M), lysine (R97K)
and aspartate (E187D). The effect of pH on the activity of
the recombinant enzymes were then determined.
Materials and methods
Materials
All reagents, unless otherwise specified, were purchased
from Sigma or Merck.
Site-directed mutagenesis
Site-directed mutagenesis was performed using as template
the plasmid pT7-7 [15] containing as insert a DNA fragment
that encodes the mature Sfbgly50 (pT7b50) [9]. The
experiments were carried out following the instructions of
the QuikChange site-directed mutagenesis kit (Stratagene).
Primers used were: R97K, 5¢-GCCTGGACGCTTACA
AGTTCTCCCTCTCCTG-3¢ and 5¢-CAGGAGAGGGA
GAACTTGTAAGCGTCCAGGC-3¢; R97M, 5¢-GCCTG
GACGCTTACATGTTCTCCCTCTCCTG-3¢ and 5¢-CA
GGAGAGGGAGAACATGTAAGCGTCCAGGC-3¢;
Y331F, 5¢-GATCGGAGTGAACCACTTCACAGCATT
CCTGGTATC-3¢ and 5¢-GATACCAGGAATGCTGTG
AAGTGGTTCACTCCGATC-3¢; E187D, 5¢-GTTCATC
ACTTTCAACGATCCTAGAGAGATTTGCTTTGAG-3¢
and 5¢-CTCAAAGCAAATCTCTCTAGGATCGTTGA
AAGTGATGAAC-3¢. Codons in bold show the mutations.
The incorporation of the mutated codon in the pT7b50

Hepes buffer
pH 7.0 containing 150 m
M
NaCl, 0.02% (w/v) lysozyme
(chicken egg white) and 0.1% (v/v) Triton X-100. The
suspension was incubated at 4 °C with slow shaking
(3 r.p.m.). After 30 min, the cells in the suspension were
disrupted using a sonicator adapted with a micro tip (five
pulses of 30 s at output 4 in a Branson 250 sonicator) and
Fig. 1. Schematic representation of the Sfbgly50 active site. E399 is the
nucleophile and E187 is the proton donor. Y331 and R97 form
hydrogen bonds (dotted lines) with E399 (Y331 O
g
atom to E399 O
e1
atom ¼ 2.69 A
˚
and R97 N
g1
atom to E399 O
e2
atom ¼ 2.77 A
˚
). The
distance between E399 and E187 side chains is 4.5 A
˚
.
Ó FEBS 2003 Mechanism of pH optimum control in b-glycosidases (Eur. J. Biochem. 270) 4867
harvested at 7000 g for 20 min at 4 °C. The supernatant
was stored at )20 °C and used as source of recombinant

anoside) as substrate [18]. Fractions containing b-glycosi-
dase activity were pooled and dialysed in 20 m
M
triethanolamine buffer pH 8.0, and the dialysed material
was loaded onto a Resource Q column (Amersham Bio-
science). Nonretained proteins were washed out with 20 m
M
triethanolamine buffer pH 8.0 and retained proteins were
eluted using a gradient of NaCl prepared in the same initial
buffer. The presence of the recombinant Sfbgly50 was
detected as above and its purity ascertained by SDS/PAGE
followed by silver staining [19].
Protein determinations were performed spectrophotomet-
rically (absorbance in 280 nm) using e
280
¼ 117 200
M
)1
Æcm
)1
[20]. The same protocol was used to purify the
wild-type and mutant Sfbgly50.
The native Sfbgly50 was purified from the S. frugiperda
midgut following the procedures described previously [21].
Kinetic analysis
All assays were performed at 30 °Cin50m
M
citrate/
phosphate buffer pH 6.0 and initial rate data measured.
Hydrolysis of MUbglc (4-methylumbelliferyl b-

phosphate buffer pH 8 at 30 °C. In
this pH phenylglyoxal reacts specifically with arginine
residues [23,24]. Wild-type (0.49 l
M
)ormutantSfbgly50
(0.13 l
M
) samples were incubated with the modifying
agent in the absence or presence of high concentration of
NPbglc (> 4 · K
m
). Samples were collected after differ-
ent periods of time and 10 m
M
arginine in 20 m
M
phosphate pH 8.0 was added. This material was used to
determine the remaining enzymatic activity using 4 m
M
MUbglc as substrate in 50 m
M
citrate/phosphate buffer
pH 6.0. Then, the rate constants (k
obs
)fortheSfbgly50
inactivation in different phenylglyoxal concentrations were
calculated.
pH effect on the Sfbgly50 activity
Sfbgly50 enzymatic activity on 8 m
M

a
s in the enzyme–substrate (ES)
complex of the catalytically active groups of Sfbgly50
(pK
ES1
and pK
ES2
) were determined by fitting the V
max app
of the MUbglc hydrolysis at each pH to Eqn (1) [25].
V
max app
¼
1
1 þ
H
þ
ÂÃ
K
ES1
þ
K
ES2
H
þ
ÂÃ

ð1Þ
V
max app

¼
1
1 þ
H
þ
ÂÃ
K
E1

þ
K
E2
H
þ
ÂÃ

ð2Þ
k
cat
/K
mapp
is the relative k
cat
/K
m
determined at each pH, [H
+
]
is the proton concentration and K
E1

points above the pH optimum were obtained. However, it
was not possible to go below pH 5.0 because Sfbgly50
becomes unstable.
Homology modelling
The three-dimensional structure of Sfbgly50 was modelled
according to structural data for Bacillus polymyxa
b-glucosidase A (1BGA, 1BGG, 1TR1), Trifolium repens
b-glucosidase 2 (1CBG) and Lactococcus lactis 6-phospho
b-galactosidase (1PBG). Modelling was performed in the
Swiss Model server and the result was visualized by
PDBVIEWER
[26].
4868 S. R. Marana et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Sequence alignment and structural comparison
Amino acid sequences of family 1 b-glycosidases were
retrieved from the CAZy website [1] and aligned using the
software
CLUSTALX
[27]. The spatial coordinates of family 1
b-glycosidases were retrieved from the PDB website and
visualized by
PDBVIEWER
[26].
Results
The expression of recombinant wild-type and mutant
Sfbgly50 was checked by SDS/PAGE (Fig. 2A). Recom-
binant Sfbgly50 was purified by a combination of hydro-
phobic and anion-exchange chromatography (Fig. 2B),
resulting in a 50% recovery and a yield of about 0.2 mg
purified Sfbgly50 from 0.5 L bacterial culture (Fig. 2C).

optimum (5.8) lower than the recombinant wild-type
enzyme (Fig. 5C,D).
Fig. 2. Induction and purification of the recombinant Sfbgly50. (A)
SDS/PAGE of proteins from NovablueDE3 cells transformed with
plasmid pT7-7 containing Sfbgly50. Lane 1, Noninduced cells; lanes 2,
3, 4 and 5, cells induced to produce the mutants R97M, R97K, Y331F
and E187D, respectively. The arrow indicates the recombinant
Sfbgly50. The gel (10% polyacrylamide) was stained with Coomassie
blue R. (B) The soluble material from the bacteria producing the
R97M Sfbgly50 was loaded onto a Phenyl Superose HR 10/10 column
eluted with a decreasing gradient of (NH
4
)
2
SO
4
,preparedin50m
M
phosphate buffer pH 7.0. b-Glycosidase activity (r) was detected
using 2 m
M
NPbglc prepared in 50 m
M
citrate/phosphate buffer
pH 6.0. The two most active fractions were pooled. (C) Ion-exchange
chromatography in Resource Q column of the b-glycosidase activity
recovered in (B). Elution produced using a gradient of NaCl prepared
in 20 m
M
triethanolamine buffer pH 8.0. b-Glycosidase activity (r)

family 1 b-glycosidases showed that R97 and Y331 are
totally conserved and that these residues plus the nucleo-
phile (E399) have the same spatial positioning (Fig. 6).
Nevertheless, the distance between arginine and glutamate
varies from 2.59 to 3.64 A
˚
and the distance between the
tyrosine and glutamate varies from 2.59 to 2.98 A
˚
.
Discussion
An inspection of the three-dimensional structure of some
family 1 b-glycosidases [10–13] and of the structural model
of the Sfbgly50 active site show that an arginine (R97) and a
tyrosine (Y331) are very close (2.69 A
˚
and 2.77 A
˚
, respect-
ively) and form hydrogen bonds with the side chain of E399.
The hydrogen bond between R97 and E399 probably has a
strong electrostatic component. However, determination of
the relative contribution of each component in this inter-
action is not simple. On the other hand, E399 is also close
to E187 side chain (4.5 A
˚
) and these residues may interact
electrostatically (Fig. 1). These noncovalent interactions
may modulate the E187 and E399 ionization state and
consequently determine the Sfbgly50 pH optimum. In order

(m
M
)
k
cat
(s
)1
)
k
cat
/K
m
(s
)1
Æm
M
)1
)
k
cat
/K
m
relative
Wild-type 2.3 ± 0.1 1.73 ± 0.09 0.75 ± 0.06 100
R97M 1.9 ± 0.3 0.0030 ± 0.0002 0.0015 ± 0.0005 0.2
Y331F 2.0 ± 0.5 0.0070 ± 0.0005 0.003 ± 0.001 0.45
E187D 4.4 ± 0.1 0.00147 ± 0.00002 0.00033 ± 0.00001 0.044
Fig. 3. Effect of pH on the activity of native (j)andrecombinant(s)
wild-type Sfbgly50. The buffers used were 50 m
M

MUbglc in 50 m
M
citrate/phosphate pH 6.0.
4870 S. R. Marana et al. (Eur. J. Biochem. 270) Ó FEBS 2003
The influence of R97 on catalysis is confirmed by the
phenylglyoxal inactivation (Fig. 4), which is abolished by
substrate and is not observed with the R97M and R97K
mutants. The reaction order relative to phenylglyoxal (1.7)
indicates that the enzyme is inactivated by the reaction of
two phenylglyoxal molecules with one arginine residue.
Despite the fact that the reaction mechanism is not clear (a
dimer or two phenylglyoxal molecules may react with one
arginine residue), the reaction order (1.7) is in agreement
with the proportion found in reactions between phenyl-
glyoxal and polypeptides (2 : 1) [23]. The structure of the
putative reaction product [23] indicates that the modified
R97 side chain is unable to hydrogen bond with E399,
probably causing wild-type Sfbgly50 inactivation. More-
over, the addition of a bulky group (diphenylglyoxal) in the
active site probably hinders substrate binding.
The substitution of R97 by M results in a 500-fold
decrease in k
cat
, whereas the replacement of Y331 by F
results in a 250-fold decrease (compare k
cat
for the wild-type
and mutant Sfbgly50 in Table 1). As the extent of k
cat
decrease is similar in both cases, R97 and Y331 may have a

by
+0.6 pH units (from 4.8 to 5.4), but had no effect on the
E187 pK
a
. As a consequence of the higher pK
a
of E399, the
mutant R97M has a pH optimum (6.5) slightly higher than
that of wild-type Sfbgly50 (6.2).
Although the introduction of a methionine residue at
position 97 could have changed the dielectric constant of the
active site, the deletion of the hydrogen bond between R97
and E399 is probably a major cause of the shift in the pK
a
of E399. Therefore, R97 facilitates the ionization of the
catalytic nucleophile by stabilizing its charged state.
Fig. 5. Effect of pH on the relative maximum velocity (V
maxapp
)ofthe
wild-type (s) and mutant Sfbgly50 (j). (A) R97K; (B) R97M; (C)
Y331F; (D) E187D. The buffers used were 50 m
M
citrate/phosphate
(pH 4.7–7.0), 50 m
M
phosphate (pH 7.0–8.0) and 50 m
M
bicine
(pH 8.0–8.5). Each point is the average of four Sfbgly50 activity
determinations using 8 m

a
)of
ionization of the groups in wild-type and mutant enzyme.
This ionization differs because of the stabilizing effect
provided by R97, which is lacking in the mutant R97M.
Hence, the DDG° is equal to the energy of the stabilizing
effect provided by R97. Thus, based on the DpK
a
of E399
between the wild-type and R97M Sfbgly50, it was calculated
that R97 contributes 3.4 ± 0.4 kJÆmol
)1
to stabilize the
charge of E399.
In the R97K mutant the pK
a
of E399 is shifted by
+0.8 pH units, whereas pK
a
of E187 changed by +0.3 pH
Fig. 6. Sequence alignment and structural comparison of family 1 glycoside hydrolases. (A) Sequence alignment of the regions containing the residue
R97 and Y331 (Sfbgly50 numbering). The aligned b-glycosidases are from Actinomyces naeslundii AAK33123.1, Agrobacterium sp. AAA220851,
Arabidopsis thaliana Q9SE50, Bacillus circulans Q03506, Bacillus polymyxa P22073, Brassica napus Q42618, Catharanthus roseus AAF28800.1,
Cavia porcellus P97265, Clostridium longisporum Q46130, Escherichia coli K12 P11988, Lactobacillus caseii P14696, Lactococcus lactis P11546,
Prunus serotina AAL06338.1, Pyrococcus woesei O52626, Sinapis alba P29092, Spodoptera frugiperda AF052729, Staphylococcus aureus P11175,
Sulfolobus solfataricus P22498, Thermus nonproteolyticus AAF36392.1, Trifolium repens P26205, Zea mays P49235. An asterisk marks identical
residues, a colon indicates strongly conserved residues and a period denotes weakly conserved residues. (B) The spatial position of the residues
corresponding to Y331, E399 and R97 (Sfbgly50 numbering) in different glycoside hydrolases was superimposed. The spatial coordinates were
retrieved from PDB: 1BGG, b-glycosidase from Bacillus polymyxa (black; R77, Y296 and E352); 2MYR, myrosinase from Sinapis alba (green;
R95, Y330 and E409); 1CBG, cyanogenic b-glycosidase from Trifolium repens (orange; R91, Y326 and E397); 1PBG, phospho b-galactosidase from

(from 4.8 to 5.5) and the pK
a
of E187 by +1.6 pH units
(7.4–9.0). As a result, the pH optimum of the Y331F mutant
(7.0) is higher than that of the wild-type Sfbgly50 (6.2)
(Fig. 5). The effect of this mutation on the E399 ionization
is the same as observed for the mutation R97M
(DpK
ES1
¼ + 0.6; Table 2), indicating that Y331 also
stabilizes the charged E
399
. Part of this effect may result
from an alteration of the dielectric constant of the active site,
although the deletion of the hydrogen bond between Y331
and E399 is probably a major component of that pK
a
increment. Based on the DpK
a
of E399 between the wild-
type and Y331F Sfbgly50 it was calculated that the Y331
contributes 4.0 ± 0.4 kJÆmol
)1
to the stabilization of the
charged E399, the same value observed for R
97
.Therefore,
R97 and Y331 together contribute 7.4 kJÆmol
)1
to stabil-

pK
a
of E399, in addition to it being closer to E187, may
have resulted in a large shift in the pK
a
of E187. Changes
in the dielectric constant due to F331 may further increase
the pK
a
of E187.
This unexpected shift in the pK
a
of E187 cannot be
directly compared with data from other b-glycosidases,
but the effect of the Y331F mutation in the pH-dependent
activity profile is very similar to that observed in the
mutant Y298F of the Agrobacterium b-glycosidase [14].
The results here obtained are similar to those found for a
family 11 xylanase interaction between a tyrosine and a
charged glutamate. The deletion of a hydrogen bond
between a tyrosine and the catalytic nucleophile (glu-
tamate)inthexylanasealsoresultedinalargeshift(+1.6
pH units) in proton donor pK
a
[29]. However, this
comparison is not strong because family 11 does not
belong to clan A [1], implying in different active site
structure and composition.
The mutation E187D also resulted in a large decrease in
k

a
. However, the pK
a
values of free aspartic and glutamic acids side chains were
determined in water, thus they do not have necessarily the
same properties in an environment hidden from the
solvent like that of the active site (less than 5% of
the E187 area is exposed to the solvent). In these
conditions the ionization of aspartic and glutamic acids
may be equally unfavourable.
Thus, if this hypothesis is correct, the DpK
a
of the
catalytic proton donor may really indicate the presence of
an electrostatic repulsion between residues E187 and E399.
This hypothesis is further supported by the results obtained
with b-glucosidase from Agrobacterium sp. (family 1) [4].
Here, the replacement of the catalytic nucleophile (E358) by
an aspartic acid resulted in a decrease of 0.9 pH units in the
pK
a
of the catalytic proton donor – a result also interpreted
as an indication of an electrostatic repulsion between the
catalytic glutamates [4]. An electrostatic repulsion between
the catalytic glutamates was already described in a xylanase
from family 11 [8], although one must be cautious with this
comparison, as noted above.
In conclusion, the combination of these results shows that
residues R97 and Y331 modulate the ionization of residue
E399 by stabilizing its charge and reducing its pK

), except in the Zea mays b-glycosidase, where the
distance between the arginine and glutamate is 3.64 A
˚
.But
even in this case, a small movement in the flexible arginine
side chain would that distance shorter without any steric
hindrance.
This structural conservation suggests that the same
noncovalent interactions are formed in all family 1
b-glycosidases. On this basis it is proposed that the noncova-
lent interactions network that modulates the Sfbgly50 pH
optimum is probably operating in all family 1 b-glycosidases.
Acknowledgements
This project is supported by FAPESP (Fundac¸ a
˜
odeAmparoa
`
Pesquisa do Estado de Sa
˜
o Paulo) and CNPq (Conselho Nacional de
Desenvolvimento Cientı
´
fico e Tecnolo
´
gico). E.H.P.A. and L.M.F.M.
are undergraduate student fellows of CNPq. S.R.M., W.R.T. and C.F.
are staff members of the Biochemistry Department (IQUSP) and
research fellows of CNPq.
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